A significant portion of currently approved drugs target G Protein Coupled Receptors (GPCRs) but most have off-target effects and many of these include dental sequelae. Such side effects can arise when distinct GPCRs in one cell signal through similar second messengers. Thus, the stimulation of one GPCR affects the down- stream signaling pathway and adaptation of the cell's other GPCRs. Indeed, one key question in the field is how different GPCRs, though they activate many of the same second messengers, signal independently without cross adaptation of their signals. Understanding how this signaling is segregated may lead to the ability to develop interventions that limit crosstalk and off-target effects. My overarching goal is to understand how adaptation of GPCR pathways can be independently regulated. This question will be studied in the C. elegans model system, where signaling from multiple GPCRs can be studied in individual olfactory neurons. Our lab recently discovered that an endogenous RNAi (endo-siRNA) pathway promotes adaptation to GPCR signaling by odorants. This observation provides a framework to explain how multiple odors sensed by a single sensory neuron are adapted to independently. This may thus constitute one mechanism by which highly similar signal transduction pathways are insulated from each other in the same cell. This leads me to the hypothesis that the specificity of adaptation to G protein signaling in olfactory neurons is directed by the engagement of different endogenous RNAi biosynthetic pathways acting downstream of olfactory GPCR stimulation. I will test this hypothesis in 3 aims: In aim 1, I will use behavioral assays to identify endogenous RNAi processing factors that act downstream of GPCRs to promote adaptation specifically to either isoamyl alcohol (IAA) or butanone (BU). Preliminary data suggests that the exonuclease ERI-1 is one such factor as it is required for adaptation only to IAA. In aim 2, I will use biochemical and visual means to determine whether ERI-1 is, in fact uniquely required for adaptation to IAA and if so, by what mechanism. In aim 3, I will use high-resolution endo-siRNA sequencing of either IAA or BU adapted populations to determine if specific siRNA species are produced in adaptation to each odor. I will then determine whether any of these sequences or removal of their targets are sufficient to promote IAA adaptation. Together, these results will provide insight into how GPCR signaling can remain separated within the same cell, and how endogenous siRNA mediates this process.